Probing interaction of human serum albumin with the tautomers of 3,4′,7,8-tetrahydroxyflavone by spectroscopic and mass spectrometric approaches

3,4′,7,8-Tetrahydroxyflavone (3,4′,7,8-tetraHF) is a kind of plant flavonol, and possesses anti-inflammatory, anti-nociceptive and anti-platelet effects. For understanding its pharmacology, the binding mechanism of 3,4′,7,8-tetraHF to a model protein, human serum albumin (HSA), was probed by fluorescence and UV absorption and mass spectrometric approaches. The binding affinities of the drug with the native HSA and its isomer were about 1.64 ± 0.54 × 105 L mol−1 and 0.68 ± 0.53 × 105 L mol−1, respectively. The UV absorption results showed that the hydroxyl groups for both the keto and enol forms of 3,4′,7,8-tetraHF were dissociated and the drug bound with the protein in anion. The fluorescence emission intensities and anisotropy of 3,4′,7,8-tetraHF were dramatically enhanced after interacting with the protein. The binding site of the drug on HSA was located on the motional restricted hydrophobic pocket of subdomain IIA, namely site I, and the drug–protein complex was mainly stabilized by electrostatic and ionic forces.